RESUMO
26S proteasomes are known as major non-lysosomal cellular machines for coordinated and specific destruction of ubiquitinylated proteins. The proteolytic activities of proteasomes are controlled by various post-translational modifications in response to environmental cues, including DNA damage. Besides proteolysis, proteasomes also associate with RNA hydrolysis and splicing. Here, we extend the functional diversity of proteasomes by showing that they also dynamically associate with microRNAs (miRNAs) both in the nucleus and cytoplasm of cells. Moreover, DNA damage induced by an anti-cancer drug, doxorubicin, alters the repertoire of proteasome-associated miRNAs, enriching the population of miRNAs that target cell cycle checkpoint regulators and DNA repair proteins. Collectively, these data uncover yet another potential mode of action for proteasomes in the cell via their dynamic association with microRNAs.
Assuntos
Dano ao DNA , MicroRNAs/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Antibióticos Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Doxorrubicina/farmacologia , Células HEK293 , Humanos , Células K562 , MicroRNAs/genética , Complexo de Endopeptidases do Proteassoma/genéticaRESUMO
The aim of this study was to determine the functional significance of peroxiredoxin V (PRXV) in defense against oxidative stress and changes of its expression in human lung inflammation. We used in vitro cell cultures and retrospective analyses of human sputum samples to perform the study. We found that stable clones of lung epithelial cell lines A549 and U1810 with reduced expression of PRXV were prone to oxidative damage. Upregulation of PRXV decreased induction of DNA double-strand breaks and protein oxidation by cigarette smoke extract and hydrogen peroxide. Transfection with PRXV-carrying plasmid protected Calu-3 confluent epithelial cell sheets from alterations in barrier permeability induced by oxidative stress. In human sputum proinflammatory cytokines, myeloperoxidase, and PRXV were increased during viral-induced inflammation. We conclude that PRXV is an important antioxidant protein of lung epithelial cells. Its expression in the human lung increases in inflammation.
Assuntos
Antioxidantes/metabolismo , Células Epiteliais/metabolismo , Pulmão/metabolismo , Peroxirredoxinas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Carcinoma Pulmonar de Células não Pequenas/patologia , Linhagem Celular Tumoral , Resfriado Comum/metabolismo , Citocinas/metabolismo , Células Epiteliais/patologia , Humanos , Pulmão/patologia , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Epiteliais e Glandulares/metabolismo , Neoplasias Epiteliais e Glandulares/patologia , Estresse Oxidativo/fisiologia , Estudos Retrospectivos , Escarro/metabolismo , TransfecçãoRESUMO
BACKGROUND: We characterized changes in expression of the antioxidant protein Peroxiredoxin V (PRXV) during airway inflammation. METHODS: Studies in anesthetized rats and mice; PRXV expression determined by Western blot analyses and immunohistochemistry; PRXV m-RNA expression determined by Taq-Man RT-PCR. RESULTS: Bacterial lung inflammation did not change expression of PRXV in murine epithelia but produced massive influx of leukocytes highly expressing PRXV. Endotoxin and f-MLP induced leukocyte migration in rat trachea but did not change mRNA levels and PRXV protein expression in tracheal epithelial cells. In primary airway cell culture (cow), alveolar epithelial cells A549, or co-culture of A549 with murine macrophages RAW264.7, exposure to live bacteria increased expression of PRXV, which required serum. PRXV was secreted in vitro by epithelial and immune cells. CONCLUSION: Inflammation increased expression of PRXV in airways by at least 2 mechanisms: cell population shift by massive influx of leukocytes expressing PRXV, and moderate post-transcriptional up-regulation of PRXV in epithelial cells.
RESUMO
Antioxidant protein Peroxiredoxin V (PrxV) is located in mitochondria and peroxisomes but is also present in the nucleus. Here, we show that nuclear PrxV associates with coilin-containing bodies suggesting possible interaction of this protein with transcription complexes. We also studied etoposide-induced phosphorylation of histone H2AX (gamma-H2AX) in human cells in which PrxV activity was downregulated (knockdown, KD-clones) or compromised by overexpression of redox-negative (RD) protein. In KD clones, but not in RD-clones, formation of etoposide-induced gamma-H2AX was increased, indicating that PrxV inhibits conversion of topoisomerase II cleavage complexes into double-strand DNA breaks but this inhibition is not caused by its antioxidant activity.